Abstract Gene transfer to, and expression in, neurons in vivo We have been developing HSV as a vector for gene transfer to the nervous system using a mouse model of a human lysosomal disease. Specifically, we have used a -glucuronidase (GUSB) negative mouse (gusmps/gusmps), with mucopolysaccharidosis (MPS) type VII (Sly disease), as an animal model. We have overcome the problem of viral toxicity in the CNS by using ICP34.5 deletion mutants (the deletion used in HSV for human cancer therapy trials) and studied the spread of, and expression from, vector genomes following inoculation at various locations in the CNS. Using the MPS VII mouse model, the long-term level of GUSB expression was found to be within the range required to correct disease. In the present application we wish to extend the utility of HSV virus vectors by developing an expression cassette utilizing the LAT promoter and the LAT gene chromatin boundaries from HSV. These elements give the LAT gene its long term expression characteristics and make it so desirable for vector expression. This cassette will enable long-term expression from our HSV vector to be improved. It will be engineered into another viral vector, to determine whether all the elements for long-term stable expression in the HSV vector are contained within the cassette. We will also answer a basic question about our HSV vector: does it need to replicate in order spread efficiently through the nervous system? The goal of our studies is to address the problem of treatment of the global lesions in neurogenetic diseases by developing a method to distribute a vector genome widely in the nervous system in vivo, using a ubiquitous human virus. These studies also provide a model for studying gene regulation in neuronal cells in vivo using state of the art techniques of molecular biology.